Genome sequencing and functional characterization of a Dictyopanus pusillus fungal extract offers a promising alternative for lignocellulose pretreatment of oil palm residues

The pretreatment of biomass is a critical requirement of bio-renewable fuel production from lignocellulose. Although current processes primarily involve chemical and physical approaches, the biological breakdown of lignin using enzymes and microorganisms is quickly becoming an interesting eco-friendly alternative to classical processes. As a result, bioprospection of wild fungi from naturally occurring lignin-rich sources remains a suitable method to uncover and isolate new species exhibiting ligninolytic activity. In this study, wild species of white rot fungi were collected from Colombian forests based on their natural wood decay ability and high capacity to secrete oxidoreductases with high affinity for phenolic polymers such as lignin. Based on high activity obtained from solid-state fermentation using a lignocellulose source from oil palm as matrix, we describe the isolation and whole-genome sequencing of Dictyopanus pusillus, a wild basidiomycete fungus exhibiting ABTS oxidation as an indication of laccase activity. Functional characterization of a crude enzymatic extract identified laccase activity as the main enzymatic contributor to fungal extracts, an observation supported by the identification of 13 putative genes encoding for homologous laccases in the genome. To the best of our knowledge, this represents the first report of an enzymatic extract exhibiting laccase activity in the Dictyopanus genera, offering means to exploit this species and its enzymes for the delignification process of lignocellulosic by-products from oil palm.


Introduction
The accumulation of agro-industry lignocellulosic postharvest by-products is a direct consequence of the global demand for crops employed in the food supply chain and bio-renewable fuel production. Following this trend, global palm oil production has increased 43% over the past 10 years to reach 52 million tons in 2015, primarily due to high biodiesel demand (1). As a result, the product-towaste ratio for palm oil production remains significantly high (1:3), generating important lignocellulosic biomass accumulation (2). This represents a particularly pressing environmental issue for the largest producing countries such as Malaysia and Indonesia. One alternative to overcome the significant build-up of cellulosic biomass is the production of bioethanol by fermentation of syrups extracted from cellulose and hemicellulose hydrolysis. Lignocellulosic ethanol production is an eco-friendly alternative to current agro-industry by-products, in addition to offering an important source of renewable energy (3).
Lignocellulose is a raw material composed of lignin, cellulose, and hemicellulose, forming a complex aromatic polymer that provides rigidity and strength to plant cell walls. While cellulose represents an inestimable carbon energy source on a global scale, releasing cellulose from lignocellulose by lignin removal represents a major challenge in many industrial processes, including the bioethanol and pulp and paper industries (4)(5)(6). To this day, delignification is either performed by chemical strategies using environmentally damaging acids or alkaline solutions, and/or through physical processes such as high temperature and pressure conditions (7). A biological delignification process using ligninolytic enzymes that breakdown lignin through an oxidation mechanism would therefore offer a valuable alternative for the pretreatment of lignocellulose (8). Laccases (EC 1. 10.3.2), manganese peroxidases (EC 1.11.1.13), and lignin peroxidases (EC 1.11.1.14) are the most promising ligninolytic catalysts for such biological pretreatment. These enzymes are primarily expressed and secreted from basidiomycete fungi, especially the Agaricomycetes class (9). Fungi are the main organisms associated to wood decay colonization due to their ability to secrete oxidoreductases and their high affinity for phenolic polymers such as lignin.
Studies on fungi lignocellulose decomposition have thus demonstrated that species involved in wood decay produce a pool of many enzymes acting against the three primary lignocellulose components (10,11).
It has been established that co-evolution between white-rot fungi and angiosperms favored the specialization of ligninolytic enzymes to degrade lignin and a broad range of compounds derived from wood decay, turning these organisms into valuable biotechnological tools (12,13). Fungi enzymatic extracts exhibiting ligninolytic activities are thus currently positioned as a promising biotechnological tool for the management of recalcitrant pollutants such as dyes, pesticides, phenolic compounds, and agroindustry residues (14,15). Nevertheless, fungus-based lignocellulosic pretreatment processes for industrial applications is still hampered by the difficulty to produce large amounts of highly active enzymes. Luckily, these problems can partly be overcome by the use of recombinant organisms and/or screening of species with enhanced enzymatic ability (16,17). Additionally, new sequencing techniques used in combination with fungi bioprospecting can improve our understanding of the enzymatic delignification process performed by fungi during lignocellulose recycling. Such knowledge can then serve as basis to develop biotechnological alternatives to handle lignocellulosic residues from agro-industry, potentially leading to new developments in the production of bioethanol and/or organic compounds (18)(19)(20).
Herein, we describe the isolation, whole-genome sequencing of D. pusillus, and initial characterization of wild basidiomycete enzymatic extracts exhibiting ABTS oxidation as an indication of laccase activity. To shed light on potential enzymes involved in this ligninolytic activity, the genome of D.
pusillus was sequenced using single-molecule real-time sequencing technology, de novo assembled, and annotated. Our main goal was to identify new fungal enzymatic tools capable of sustaining harsh experimental conditions for extended periods of time, such as higher temperatures and lower pH, while favoring an increase in the release of reducing sugars during simultaneous pretreatment and saccharification (SPS) processes of empty fruit bunch from oil palm trees. We found that laccase activity was the main enzymatic contributor to our fungal extracts, which included a highly active isolate from D. pusillus LMB4. In addition to characterizing potentially valuable biotechnological tools for the enzymatic lignocellulose pretreatment of palm tree residues, our results also present the first complete genome sequencing of a Dictyopanus fungus.

Fungi isolation and growth conditions.
Fruit bodies from basidiomycete fungi growing on decaying wood were collected in a tropical humid forest in Colombia, following previously published parameters to favor the presence of delignification enzymes (21,22). The main inclusion criteria were macroscopic properties belonging to the orders of Agaricales, Russulales, and Polyporales due to the possible ligninolytic activity of these organisms (23,24). Collected samples were kept in wax paper bags to prevent deterioration. Isolation of the collected fungi was performed in wheat bran extract agar composed of 18 g.L -1 agar, 10 g.L -1 glucose, 5 g.L -1 peptone, 2 g.L -1 yeast extract, 0.1 g.L -1 KH 2 PO 4 , 0.1 g.L -1 MgSO 4 .7H 2 O, 0.085 g.L -1 MnSO 4 , 1000 mL wheat bran extract, 0.1 g.L -1 chloramphenicol, 0.1 g.L -1 and 600 U.L -1 nystatin. Pilei were adhered to the top cover of Petri dishes, allowing spores to fall and, eventually, to germinate on the culture media. Top covers were rotated every 24 h for 3 days and those containing the pilei were replaced by new sterilized ones (25). Sub-cultures in the same media were incubated at 25°C to obtain axenic strains from these isolates. The axenic cultures were determined by fungal slide culture technique (26). The presence of microscopic sexual basidiomycete properties was checked, including septate hyaline hyphae and clamps. Lactophenol cotton blue stain was used for all the microscopic observations. Twelve ligninolytic fungi belonging to genera Aleurodiscus, Dictyopanus, Hyphodontia, Mycoacia, Phellinus, Pleurotus, Stereum, Trametes, and Tyromyces were axenically

Phylogenetical identification of selected isolates.
Total genomic DNA was extracted from selected isolates following a standard phenol-chloroform protocol. Briefly, fungi were grown in wheat bran extract agar for 15 days and 0.5 g of mycelium was placed in a tube with a lysis solution (0.1 M NaCl 2 , Tris-HCl pH 8, 5% SDS) and 0.5 mm diameter glass beads. The aqueous fraction was collected, and the fungus DNA was precipitated with isopropanol. The DNA pellet was dissolved in TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0) (27). A pair of primers within the Internal Transcribed Spacer regions (ITS1/ITS4) was used to amplify ribosomal DNA by PCR (28). PCR products were sequenced by the Sanger method using the same amplification primers. ITS1 sequences were used as query to retrieve the most similar DNA sequences from the NCBI database. A set of 36 curated sequences were extracted from the results obtained through BLAST, after which the ITS1 sequences and the query sequence were used to create a multiple sequence alignment. To infer the evolutionary history and obtain the genetic identity of the fungus isolated and pre-identified as Dictyopanus sp., we applied the UPGMA protocol, where the best tree hits arose after a bootstrap of 500 repetitions using the Maximum Composite Likelihood method to obtain the evolutionary distances. All phylogenetic analysis were performed with the MEGA suite, version 10.0.5 (29).

Fiber analysis of oil palm by-products. Neutral Detergent Fiber (NDF), Acid Detergent
Fiber (ADF), and Acid Detergent Lignin (ADL) were determined by the Van Soest method using the FiberCap TM system (Foss Analytical AB, Denmark). Cellulose and hemicellulose percentages were estimated as the difference between ADF and ADL, and NDF and ADF respectively, while lignin concentrations corresponded to ADL percentages in dry weight of oil palm by-products. Additionally, values were used to estimate the total carbon concentration in fermentation assays. All assays were performed in duplicate.

Basidiomycete screening by solid-state fermentation (SSF). The main selection criterion
of isolated wild-type fungi was ligninolytic activity observed in the crude extracts from SSF using lignocellulosic material from oil palm by-products (30). SSF was performed in 250 ml flasks in sterile conditions. Each flask contained 12 ml of basal media in deionized water, comprising 0. ZnSO 4 ·7H 2 O, 0.0037 g.L -1 CoCl 2 ·6H 2 O, and 2.5 g.L -1 of empty fruit bunch (EFB) chopped into chunks of approximately 2 cm 3 . Each flask was inoculated with eight agar plugs cut from actively growing fungal mycelium grown on wheat bran extract agar. Each SSF batch isolation contained thirty flasks and fermentation was held without agitation at 25˚C for 30 days. Every three days, three flasks were used to collect crude enzymatic extracts.

Recovery of crude enzymatic extracts.
Crude enzymatic extracts were obtained by addition of 30 ml of 60 mM sterile phosphate buffer into the fermentation flask, which was shaken for 24h at 150 rpm. Whole flask contents were then collected in 50 mL tubes, vortexed in a Benchmark Scientific multi-tube vortexer for 15 minutes at 1500 rpm, and finally centrifuged twice at 8900g for 15 minutes to remove suspended solids. Supernatants were taken as crude enzymatic extracts (31) and concentrated by lyophilization to evaluate the effects of pH and temperature on enzymatic activity and the SPS.

Quantification of reducing sugars.
Reducing sugars were quantified by oxidation of 3,5dinitrosalicylic acid to 3-amino,5-nitrosalicylic acid (DNS) by measuring the release of the reducing extremity of sugars. The reaction was followed at 420 nm and a standard curve was obtained with glucose (0,1 to 1 mg.ml -1 ) to quantify the concentration of reducing sugars (32).

Ligninolytic and cellulase assays.
Crude enzymatic extracts obtained from SSF were assayed for laccase, lignin peroxidase, and manganese peroxidase activities. Laccase activity was followed by the oxidation of 2,2′-azino-bis The total cellulosic activity was quantified by units of paper filter (UPF.ml -1 ). In tubes, 500 µL of commercial cellulase solutions from Trichoderma reesei Sigma Aldrich C2730 Celluclast® (USA) were incubated with 500 µL of 50 mM citrate buffer at pH 4.8, 50 and 5 mg of filter paper for 1 h, at 50 °C. The concentration of reducing sugars released was measured by the oxidation of 3,5-dinitrosalicylic acid (DNS), as described above (36). Cu-oxidase Pfam domains (PF00394, PF07731, and PF07732 entries) (43). Comparisons with the Laccase and Multicopper Oxidase Engineering Database (44) was also used to validate that the identified sequences were laccases.

Fungi isolation.
From all fruit bodies collected, twelve axenic cultures were obtained and thirty one isolations exhibited fungal contamination from biota mycoparasitism associated to basidiomycetes, mainly from Trichoderma species (data not shown). These fungi possess fungicide and antagonistic activity against basidiomycete cell walls, in addition to releasing enzymes such as chitinases and glucanases (45,46). Moreover, basidiomycete recovery from collected samples can also suffer from competition with ascomycete fungi. Competition between these two fungi heavily relies on nutrient accessibility, growth factors favoring ascomycetes due to their faster growing pace in complete culture media, or even the presence of simple nutrient sources observed in advanced stages of wood decay (47).

Screening of isolates.
Enzymatic extracts were screened for enzymes known to participate in the delignification process, i.e. laccase, manganese peroxidase, and lignin peroxidases. From the crude enzymatic extracts obtained by SSF, only five isolates exhibited laccase activity in our screening assay. Surprisingly, we were unable to measure peroxidase activity other than through the ABTS assay.
Since peroxidases are common enzymes present during fungi-catalyzed wood decay, peroxidase activity was either negligible in our isolates or the enzymatic assay was not sensitive enough to quantify such activity. It has been reported that variations in the concentrations of lignin, carbon, nitrogen, and the presence of chemical compounds such as inducers in the culture media could affect the profile of ligninolytic enzymes expressed and secreted during fermentation (51)(52)(53). While current experiments cannot explain whether the lack of peroxidase activity is related to the composition of the culture media, the abovementioned results confirm previous reports suggesting that laccase activity is the most prevalent ligninolytic activity observed during fermentation with lignocellulose as substrate (54,55).
Isolates exhibiting ligninolytic activity were identified as Dictyopanus sp. LMB4 (22.3 U.L -1 ), Pleurotus sp. LMB2 (69.5 U.L -1 ), and Pleurotus sp. LMB3 (57.2 U.L -1 ) (Fig. 1). Laccase activity of the Hyphodontia and Trametes isolates was considered too low to warrant further characterization. For the three most active isolates, the highest laccase activity was detected after 20 days of fermentation. Using these 3 isolates, laccase activity conditions were optimized by increasing copper concentration and carbon-to-nitrogen ratios (C/N) (56,57). As a result, the isolate exhibiting the highest laccase activity under these newly optimized conditions was Dictyopanus sp. LMB4 (267.6 U.L -1 after 28 days of fermentation). To the best of our knowledge, this represents the first observation of significant laccase activity in a crude enzymatic extract from a Dictyopanus fungus. Furthermore, this activity is similar to a previously reported Trametes sp. laccase activity evaluated under comparable fermentation conditions using lignocellulosic by-products from oil palm (218.6 U.L -1 ) (58). The maximal laccase activities of the Pleurotus isolates were at least 5 times lower than the one observed in Dictyopanus sp. LMB4, with 98 U.L -1 for Pleurotus sp. LMB2, and 66.9 U.L -1 for Pleurotus sp. LMB3 (Fig. 1).

Figure 1.
Laccase activity of SSF isolates. ABTS oxidation activity was tested for three culture supernatants from Dictyopanus LMB4 (circles), Pleurotus LMB2 (squares), and Pleurotus LMB3 (triangles) isolates. With a C/N ratio of 1.9 and in the absence copper, the Pleurotus spp. isolate exhibited the highest laccase activity (see inset). However, a 12-fold increase in laccase activity was observed in the Dyctiopanus sp. isolate with a 10-fold increase in the carbon-to-nitrogen ratio (19 C/N) and 5 mM copper (main histogram). Axes and units are the same for both histograms. The Dictyopanus LMB4 isolate is highlighted by an asterisk in both histograms.
Upon growth condition optimization, the crude enzymatic activity of Dictyopanus sp. LMB4 increased 6-and 12-fold after 20-and 28-day incubation, respectively, highlighting the importance of copper and carbon source accessibility for proper enzyme expression. The increase in laccase activity for enzymatic extracts upon copper and glucose addition has been reported for Colorios versicolor and Ganoderma lucidum. These reports suggested that copper and glucose could respectively stimulate laccase expression and mycelial growth, further correlating with a proportional increase in the amount of laccase secreted by the fungi (59,60). For the enzymatic extract of D. pusillus, the calculated laccase activity obtained per gram of oil palm by-products was 31.5 U.g -1 after 12 days of SSF. It is worth mentioning that this activity is four times higher than the previously reported laccase activity of a Pycnoporus sanguineus enzymatic extract obtained under similar SSF conditions using EFB as substrate (7.5 U.g -1 ) (61).

Molecular identification of Dictyopanus sp.
In contrast to most organisms genetically identified using 16S ribosomal RNA sequencing, using Internal Transcribed Spacer regions (ITS) is considered a more appropriate method to identify species in the fungi kingdom (62). In the past, mycologists have used an arbitrary sequence similarity cutoff ranging between 3-5% ITS identity as a threshold for species differentiation. However, the natural variability of ITS sequences at the phylum level within the fungi kingdom complicates the use of such cutoff (62). For instance, in Basidiomycota (to which the Dictyopanus genus belongs), the infraspecific ITS variability was reported to average at 3.3%, with a standard deviation of 5.62% (62). This significantly limits the use of GenBank BLAST searches as the only source to properly identify fungi species, especially considering the fact that more than 27% of ITS sequences were submitted with insufficient taxonomic identification (63). In addition, until 2003, nearly 20% of all fungal species listed in GenBank were incorrectly annotated (64). As a result, using BLAST searches to categorize fungal species can lead to serious misidentification and characterization.
Consequently, fungal specimen identification requires a careful, systematic, and multi-source process.
To overcome some of these limitations, we first performed preliminary in situ morphological identification of the samples collected in the Colombian forest. Genus level inspection was performed in the laboratory using macroscopic and microscopic examination, followed by final phylogenetic identification through DNA extraction and sequencing of ITS regions 1 and 4 (28). This allowed identification of the pusillus species, to which the Dictyopanus LMB4 fungus sample belongs (Fig. 2). The same analysis also allowed us to differentiate the evolutionary history for some members of the Panellus genus, with which members of the Dictyopanus genus are often confused. Results presented in Figure 2 support the usefulness of taxonomic classification performed during fungi sample collection, selection, and isolation. The Dictyopanus genus belongs to the Agaricomycetes class, and its genus is known to include species capable of bioluminescence, which have been suggested to be linked to delignification processes through the use of secondary compounds produced during lignin degradation (65).
Dictyopanus isolates were also reported as an alternative for the pretreatment of remazol brilliant blue R (66) and bamboo in ethanol production (67), further supporting the potential use of this fungus in largescale biomass degradation.

Figure 2.
Phylogenetic analysis of the pre-identified isolates labeled as Dictyopanus sp. We used the ITS region 1 as the genetic marker to infer the evolutionary history of this fungus using the UPGMA protocol (see Materials and Methods for details). The optimal tree analysis shows a branch length of 0.60, with clustering of species after a bootstrap of 500 replicates using the Maximum Composite Likelihood method to obtain evolutionary distances between members. The species was identified as Dictyopanus pusillus.
The phylogenetic tree was drawn to use the same branch length units as those of the evolutionary distances. This analysis was performed using the standalone MEGA software, version 10.0.5.

Effect of pH and temperature on the enzymatic extracts obtained from D. pusillus.
Characterization of crude enzymatic extract isolates showed that pH values between 3 and 5 provided the highest laccase activity for D. pusillus LMB4, with a maximum activity at pH 3 (Fig. 3a). This pH range corresponds to other laccase preferences in fungi (68). Moreover, thermal stability of the crude D. pusillus LMB4 enzymatic extract was found to be quite robust, with reduced activity only observed at 60°C (46% activity loss after 6 hours of incubation). This behavior is quite different from that observed with the T.
versicolor commercial laccase under the same experimental conditions, showing 28% and 78% activity loss after a 6h incubation at 50°C and 60°C, respectively (Fig. 4). Thus, D. pusillus LMB4 appears to express laccases with enhanced thermostability and higher tolerance to lower pH values. However, long incubation of this crude enzymatic extract at low pH resulted in an important activity loss (Fig. 3b).
Previous studies have shown that a laccase from Physisporinus rivulosus remained stable at 50°C with optimal activity at pH 3.5 (69). Similarly, a laccase from Trametes trogii was shown to sustain temperatures up to 75°C, although only for short 5-min incubations (70). Nevertheless, our results suggest that the laccase activity from the D. pusillus LMB4 extract has higher tolerance to acidic and thermally induced perturbations than previously identified fungal laccases.  Reducing sugar release was observed when the cellulolytic enzymatic extract from T. reesei was used alone (20.84 ± 0.7 g.g -1 ). Higher reducing sugar release from EFB was also observed when the cellulolytic enzymatic extract from T. reesei was used with the commercial laccase enzyme from T. versicolor (46.47 ± 5.9 g.g -1 ) or the enzymatic extract from D. pusillus (44.80 ± 5.21 g.g -1 ), confirming that ligninolytic enzymes such as laccases favor cellulose hydrolysis, as previously reported (72,73). These results suggest that a combination of cellulolytic and ligninolytic enzymes enhance the release of reducing sugars. However, production of reducing sugars was not significantly different when the commercial laccase from T. versicolor or enzymatic extracts from D. pusillus were mixed with the cellulolytic enzymatic extract from T. reesei (Fig. 5).
To identify the dominant variables affecting the reducing sugar release, we compared the effects of pH, temperature, copper concentration, and laccase (U.L -1 ) or cellulase (UPF) concentration using a Plackett-Burman analysis (P value <0.05 with a confidence level of 95%) (Fig. 6). In both cases (SPS with laccase from T. versicolor and D. pusillus), pH was the dominant variable affecting activity, followed by temperature and copper concentration. Our results suggest that high pH values and temperatures (up to 45°C) promote sugar release by SPS. These results confirm what was observed in our stability experiments, where the activity of the enzymatic extract was compromised at pH values lower than 4 (Fig.   3B). These results are also in agreement with prior observations suggesting that basic pH is a desirable property for laccases used in biotechnological processes, since low pH values were linked to increased enzyme degradation (74). It is worth mentioning that cellulase is the third most important contributing factor to activity when SPS is performed with the commercial laccase (Fig. 6A), a result we do not observe with the enzymatic extract from D. pusillus (Fig. 6B). The requirement of a cellulase activity in the case of the commercial laccase are perhaps due to the combined production of ligninolytic and cellulolytic enzymes in the basidiomycete fungi during wood decay processes (75,76). Some authors have also demonstrated the efficiency of enzymatic extracts from basidiomycetes for the SSF production of ligninolytic and cellulolytic enzymes using wheat straw as substrate (77). These results further highlight the importance of D. pusillus as an efficient, accessible, and economical source of relevant biotechnological assets in the field of delignification processes.
The highest reducing sugar concentration obtained with the enzymatic extract of D. pusillus was 65.87 g.g -1 (pH 4.5, 45°C, 2:1 laccase-to-cellulase ratio). In the same conditions, reducing sugar production reached 64.13 g.g -1 for the commercial laccase from T. versicolor. These results confirm that the enzymatic extract from D. pusillus exhibits similar ligninolytic efficiency than the purified commercial laccase from T. versicolor. Additionally, EFB represent a good lignocellulose source for reducing sugar production since palm oil bunches are subjected to a first round of "sterilization" to extract oil palm fruits from the bunch, which effectively acts as a pretreatment during palm oil extraction. As a result, this initial pretreatment might improve the delignification process performed by the enzymes. Lignocellulose breakdown of EFB and EFB pulp was previously reported using the white rot fungi T. versicolor TISTR 3224, Phanerochaete chrysosporium CECT 2798, and Pleurotus ostreatus CEC20311. These fungi were also used as efficient pretreatments for lignin removal in EFB (78,79). To the best of our knowledge, only one study reported the use of enzymatic extracts with laccase activity from Pycnoporus sanguineus UPM4 as a pretreatment of EFB to increase production of reducing sugars in similar conditions (80). This report and the results presented here on the use of a crude enzymatic extract exhibiting laccase activity from a white-rot fungi reinforce the relevance of using ligninolytic enzymatic extracts as a valuable tool for the pretreatment of lignocellulose in EFB.   (Table 1). After splicing of the 95,174 annotated introns, a total of 16,866 coding sequences (CDSs) were predicted to be encoded in the genome of D.
pusillus LMB4. Of this number, we confidently annotated a total of 13 CDSs as complete putative laccase sequences, which were further aligned with a previously reported laccase homolog from Trametes to identify consensus regions and conserved motifs (Fig. 7, Table S1). Our results show that all putative laccases encoded in the D. pusillus genome preserve the four conserved copper-binding motifs normally observed in this enzyme family, i.e. Cu1 (HWHGFFQ), Cu2 (HSHLSTQ), Cu3 (HPFHLHG), and Cu4 (HCHIDFHL) (41). These make them potentially promising candidates for future functional investigation of new laccases exhibiting interesting properties with respect to activity, stability, and industrial tolerance.  Consensus sequence is presented on the bottom of the alignment. Putative laccase genes are identified as in Table S1.

Conclusion
The present work demonstrates that a crude enzymatic extract from a wild Colombian source of D. pusillus LMB4 exhibits significant laccase activity (267 ± 18 U.L -1 ). This crude enzymatic extract was probed for the successful pretreatment of low-cost lignocellulosic raw materials (oil palm by-products), suggesting that an upscaling of this process could potentially help with the delignification of starting materials in cellulosic bioethanol production. An increase in copper and glucose concentration during solid-state fermentation proved beneficial, resulting in a 12-fold increase in laccase activity and suggesting that ligninolytic enzyme expression can further be induced to improve enzyme production in D. pusillus LMB4. The SPS of EFB also illustrated that the enzymatic extract from D. pusillus exhibits good ligninolytic capacity at basic pH, in addition to demonstrating higher pH and thermal stability than the purified commercial laccase from T. versicolor. These properties demonstrate the efficiency of such crude enzymatic extract from D. pusillus as a versatile biotechnological tool for lignocellulose pretreatment such as for cellulosic bioethanol production. Genome sequencing of D. pusillus LMB4 also revealed 13 laccases and a significant number of other putative enzymes that could be exploited and/or engineered to develop more efficient delignification pre-treatments. These results thus present the first few stages in the implementation of a strategy that combines genome data mining and computational modelling as efficient approaches to identify promising new protein engineering candidates as a new set of catalysts with applications in delignification processes.